Our prior studies revealed that following either needle or sand fly inoculation of L. major into the skin, neutrophils are rapidly recruited to the injection site and represent the first cell type infected. We investigated whether dendritic cells (DCs) might sense and interact with PMN in the skin, and how this contact might modulate their function as APCs. Neutrophil depletion resulted in a striking enhancement of the priming of OT-I cells following needle injection or sand fly transmission of L. major expressing OVA. Using lysozyme-GFP mice injected with RFP-expressing L. major to obtain sort-purified, infected and uninfected neutrophils, there was preferential uptake of the parasitized neutrophils by DC following their reinjection in the ear dermis. When RFP-L. major were injected directly, the majority of the infected, RFP+ DC recovered from the skin were found to harbor markers of PMN, suggesting that the normal early encounter of DC with L. major is in the context of infected neutrophils. The expression of maturation markers on infected DCs from neutrophil depleted mice was more pronounced than infected DCs from undepleted mice. Furthermore, using L. major expressing both RFP and OVA, the infected DCs from depleted mice were more efficient at activating OT-I cells ex vivo. These data demonstrate both a novel mechanism by which Leishmania parasites can induce immune dysregulation and a previously unrecognized role of infected PMN during the immune response to L. major. Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector-transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is leishmanization, in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed recruitment of protective cells and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania. Control of infection caused by Leishmania major requires the development of IFN-&#947;+CD4+ lymphocytes for the induction of microbicidal activity in host macrophages. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFN-&#947;response by the host. Susceptibility was caused by antigen-specific IL-10 from CD4+ cells that were also producing IFN-&#947;. In the present studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in Leishmaniasis. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R deficient (WSX-1-/-) mice revealed diminished IL-10 from IFN-&#947;+ CD4+ cells, which was accompanied by a reduction in total IFN-&#947;+ CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the shift in the Th1/Th2 balance and to residual levels of IL-10. Strikingly, infected WSX-1-/- mice developed more severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology. IL-10 has also been implicated in the suppression of antigen-specific T cell responses in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of L. donovani in murine models of VL. Our previous data implicated IL-10 producing, CD25-Foxp3- T cells (Tr1 cells) as the source of elevated IL-10 in the VL spleen. IL-27, a member of the IL-6/IL-12 cytokine family, was recently found to induce Th1 cell differentiation and IL-10 production, and to counter-regulate Th17 lineage development. We investigated genes related with IL-27 and Th-17 induction and function. Quantitative measurement of splenic mRNA at pretreatment showed significantly elevated levels of IL-27, IL-10, IFN-&#947;, IL-1b, IRF-1 compared to paired post-treatment samples. At 2-4 weeks post-treatment, levels comparable to healthy donor controls had been restored. By contrast, slightly elevated transcripts for IL-17 and IL-23, and significant increment in the Th17 transcription factor ROR&#947;T, was observed in the post-treatment samples. Furthermore, IL-27 protein was significantly elevated in the plasma of KA patients, which following treatment dropped to levels comparable to endemic controls. The elevated expression of IL-27 / IL-10 and low RoR&#947;T / IL-17 in host cells at pre-treatment and the reversal of this trend following clinical cure highlights the possibility that IL-27 is a regulatory cytokine central to the pathogenesis of VL.